1. Field of the Invention
This invention relates generally to the subject matter of antibodies. More particularly, it relates to monoclonal antibodies to cardiac glycoside, digoxin. The antibodies may also have cross-reactivity with digitoxin.
2. Description of the Prior Art
Digoxin and digitoxin are two of a large group of cardiac glycosides, frequently prescribed for patients with congestive heart failure. Cardiac glycosides are composed of a steroid or aglycone portion and from one to four sugar molecules. The pharmacological activity resides in the aglycone portion of the molecule and the glycoside residues modify water solubility and potency. One of the pharmacodynamic properties of this class of compounds is to increase the force of myocardial contraction.
Digoxin is an uncharged, chemically well defined hapten, sufficient in size to occupy most of the antibody combining site. For this reason, cardiac glycosides which include several structurally related analogs are suitable for the study of antibody fine specificity. But, the rigid four ring structure common among all cardiac glycosides and other steroids does not allow for major conformational changes as functional groups are substituted at various positions on the rigid steroid backbone. However, through an analysis of antibody affinity for related glycosides, it may be possible to determine antigenic determinants specific for a given anti-digoxin antibody. Certain heterogeneous antidigoxin antibodies produced in sheep have been shown to be very specific. They can discriminate between digoxin and digitoxin, which differ by the presence or absence of only a single hydroxyl group on the steroid backbone.
The possibility of reversing digoxin or digitoxin toxicity and correlating variation in the antibody combining site with differences in specificity for closely related haptens prompted the discovery of a source of unlimited supply of monoclonal antidigoxin antibodies. The successful innovation and perfection, for the first time, of the new technique of somatic cell fusion, for the production of an unlimited supply of high-affinity monoclonal antidigoxin antibodies which may also have cross-reactivity with digitoxin now opens the possibility of using such antibodies for the study of antigen-antibody structure-function relationships as well as for diagnostic and therapeutic purposes. Such a technique for the production of antibodies against digoxin has heretofore been unknown in the art.
Among various problems encountered in the somatic cell fusion technology, a major dificulty is the stability of the resulting hybridoma. Prediction, a priori, of hybridoma stability is virtually impossible. Usually after a few months of growth, the hybrids lose their specific function. The reason for this event is not yet known but mutational changes, loss of chromosome or overgrowth because of persistent multiclonality may be some of the factors for the loss of the functional activity of the hybrids. The present invention has overcome such problems and provides a method of producing stable hybridoma capable of secreting monoclonal antidigoxin antibodies. A clone of such a hybridoma, viz., Dig. 35-20, has now been prepared and deposited with the American type Culture Collection, Rockville, Md. and Identified as HB-8113 which culture is incorporated herein by reference.